Control of Eukaryotic Gene Expression (Learning Objectives)

1. Compare and contrast chromatin and chromosome: composition, proteins involved and level of packing. Explain the structure and function of nucleosome, histones, scaffold proteins (metaphase chromosomes)

2. Explain the role of chemical modifications: methylation of DNA and acetylation of histones in control of gene expression. Define the term epigenetics.

3. Identify the main mechanism for turning on gene expression. Explain why control of gene expression in eukaryotic cells is like a “dimmer switch”, an “ON” switch that can be fine tuned.

4. Identify the major switch and all the fine-tuning steps that can modulate eukaryotic gene expression.

5. Identify and explain component of eukaryotic genes: coding and regulatory sequences (proximal and distal elements)

6. Compare and contrast pre and post transcriptional and translational controls of gene expression

7. Explain interference RNA and its role play in post-transcriptional and translational regulation of gene expression

8. Define ubiquitin and proteosome and explain their roles in intracellular protein degradation

Control of Eukaryotic Gene Expression

Control of Eukaryotic Gene Expression

• Cells express 3-5% of their genes – House-keeping genes- all the time

– Genes turned on or off- internal and external signals (environmental- nurture)

– Genes turned on only in some cell types not others while others are permanently shut down

(Highly specialized nerves or muscle)

– Nucleosomal beads of chromatin: DNA and histones “Beads on a string”

– Chromatin packing is the degree of nucleosome coiling – Histones play major role in gene expression

Expose DNA when it is to be transcribed shield it when it is to be silenced

The human genome codes for ~21,000 genes

Structural organization of Eukaryotic DNA

Interphase Chromatin states

Heterochromatin: compacted,

transcriptionally inactive

Euchromatin: loosely packed,

actively transcribed

Eukaryotic Chromatin Structure Successive levels of chromatin packing 1. ds-DNA helix (naked DNA) 2. Nucleosomes (histones) 3. 30 nm chromatin 4. Looped domains (non-histone protein scaffold)

300 nm 5. Condensed scaffolded looped domains (700

nm) 6. Metaphase chromosome (1,400 nm)

Histones – Positively charged proteins

– first level of DNA packing

– Leave DNA transiently during replication

– Remain associated with DNA during transcription (change position and shape to allow RNA polymerase move along the DNA)

Packing is highly specific and precise with particular genes located in the same places

Eukaryotic gene expression

http://highered.mcgraw-

hill.com/olc/dl/120080/bio31.swf

Control of Gene Expression Nuclear level controls 1) Chromatin remodeling = “On/off” switch 2) Transcription Factors 3) Alternative splicing “Dimmer” switch includes microRNAs to

silence gene expression

Levels of control of gene expression include:

• Chromatin “remodeling” packing (Epigenetic)

• Transcription • RNA processing • RNA stability • Translation • Protein modifications • Protein degradation

Figure 11.11

Maximizing Genetic Information

Transcriptional Control

1. Chromatin-modifying enzymes - availability of DNA for transcription (On

Switch) 2. Fine-tuning begins with the interaction of

transcription factors with DNA sequences

Chromatin Remodeling Specific Chemical modifications that bind to histones and

DNA are: - Acetyl group- histones - Methyl groups- histones and DNA - Phosphate groups- histones

Histone tails

Amino acids available for chemical modification

DNA double helix

Histone tails protrude outward from a nucleosome

Acetylation of histone tails promotes loose chromatin structure that permits transcription

Unacetylated histones Acetylated histones

Chromatin Chemical Modifications a. DNA methylation

• Active genes- unmethylated (Euchromatin) • Inactive genes- highly methylated (Heterochromatin)

b. Histone acetylation

• Acetylated histones (Euchromatin) • De-Acetylated histones (Heterochromatin)

DNA methylation and histone de-acetylation

cooperate to repress transcription

Transcriptional control • Initiation of transcription- universal control of

gene expression • Controlled by interaction between proteins

and DNA

Enhancer (distal control elements)

Proximal control elements

Upstream

DNA

Promoter

Exon Intron Exon Intron Exon

Downstream Transcription

Poly-A signal sequence

Termination region

Intron Exon Intron Exon

RNA processing: Cap and tail added; introns excised and exons spliced together

Poly-A signal Cleaved 3′ end of primary transcript

3′

Poly-A tail

3′ UTR (untranslated

region)

5′ UTR (untranslated

region)

Start codon

Stop codon

Coding segment

Intron RNA

5′ Cap

mRNA

Primary RNA transcript (pre-mRNA)

5′ Exon

Proximal and Distal Control elements of gene transcription

Proximal control elements

- Control transcription initiation

- Binding sites for TATA binding protein RNA polymerase Other factors - Coordinated interaction

of a massive assembly

Distal control elements Action at a distance

- Enhancers & Silencers (Tissue specific) - Located far from the promoter upstream,

downstream, or within introns - Enhancers- bind activators - Silencers- bind repressors

Transcription and complex enhancers http://highered.mcgraw-

hill.com/sites/0072437316/student_view0/chapter18/animations.html# http://www.dnai.org/a/index.html

Distal control element Activators

Enhancer

DNA

DNA-bending protein

TATA box

Promoter Gene

General transcription factors

Group of mediator proteins

RNA polymerase II

RNA polymerase II

RNA synthesis Transcription Initiation complex

Binding of an activator to enhancer sequences bends DNA to make contact with the protein initiation complex at the promoter

Control elements

Enhancer Promoter

Albumin gene

Crystallin gene

Available activators

Available activators

Albumin gene not expressed

Albumin gene expressed

Liver cell Lens cell

Crystallin gene not expressed Crystallin gene

expressed

Liver cell nucleus

Lens cell nucleus

Role of Activators in Differential Gene Expression

Levels of control of gene expression include:

• Chromatin packing • Transcription • RNA processing • Translation • Various alterations to

the protein product • Protein degradation

Post-transcriptional control mechanisms 1. RNA processing 2. Alternate splicing 3. Half-life of RNA molecule poly A tail 5’ cap removal Nucleotide sequences in the 3’

untranslated (3’-UTR) trailer region 4. RNA interference (micro RNA)

Alternative RNA splicing Regulates coding sequence of mRNA

Primary RNA transcript

DNA

or

Exons

RNA splicing

mRNA

MicroRNAs : class of noncoding RNAs 21-22 bases long The human genome has about 1,000 distinct microRNAs

that regulate at least 1/3rd of the protein-encoding genes When a microRNA binds to a “target” mRNA, it prevents

translation Specific degradation of an mRNA Specific blocking of translation

MicroRNAs

Figure 11.7

RNA interference (RNAi) - Mechanism for silencing gene expression using

RNA molecules called RNA interference (RNAi) - Small synthetic, double-stranded RNA molecules

are introduced into selected cells to block gene expression

Dicer

Hydrogen bond

Protein complex

miRNA Target mRNA

Degradation of mRNA

OR

Blockage of translation

Translational control 1. Protein factors required to initiate 2. Specific sequences within the 5’-UTR

(leader region) of mRNA bind to regulatory proteins preventing translation

3. miRNA

Post-translational modifications Functional proteins - enzymatic cleavage - chemical modifications - transport to the appropriate destination Improperly modified proteins are promptly

degraded

Normal proteins undergo selective degradation to limit half-life

• marked by ubiquitin proteins (76 amino acids) • Giant proteosomes recognize the ubiquitin and

degrade the tagged protein http://bioisolutions.blogspot.com/2007/05/proteasomes.html